Nitric oxide synthases (NOS), the enzymes responsible for nitric oxide (NO) production from the substrate L-arginine, are also NADPH oxidases. In cell-free systems, some of these enzymes have been shown to produce reactive oxygen species such as superoxide. In this investigation, we transfected monoblastoid U937 cells with human endothelial NOS (eNOS) and found that TNFa production was increased, but that this effect was not related to NO production. Further work found that eNOS upregulates TNFa by producing a reactive oxygen species (ROS), (J Biol Chem, 2000). Recent experiments have demonstrated that eNOS upregulation of TNFa occurs through superoxide-dependent activation of p44/42 mitogen activated protein kinase (Abstract, FASEB J, 1999). Future work will focus on the switching mechanism(s) that regulate eNOS to produce either NO or ROS. A closely related effort will examine eNOS modulation of inflammatory responses in endothelial cells and the relative roles played by NO and ROS.